Structural Characterization of Chemical Compounds Based on Their Fragmentation Rules in Sophorae Fructus by UPLC-QTOF-MS/MS

Abstract

This study aims to identify the chemical components in Sophorae Fructus, and explore the mass spectrometric cleavage rules using the UPLC-Q-TOF-MS/MS method. The main characteristic fragments of the compounds were analyzed by electrospray ionization (ESI) ion source under positive and negative ion modes. The compounds were identified by molecular formula, multistage mass spectrometry, ultraviolet spectrum, and the fragmentation patterns of standards. A total of 142 compounds were identified, including 68 flavonoids, 39 saponins, 21 organic acids, and 14 others, of which 43 components were reported from Sophora for the first time. Moreover, the mass spectrometric fragmentation rules of some identified species components were deduced, which are helpful for the structural analysis of flavonoid and saponins. This method provides a reference for the rapid identification of chemical components and is conducive to further study the pharmacodynamic material basis and action mechanism of Sophorae Fructus.

Keywords

Sophorae Fructus - flavonoid glycosides - saponins - UPLC-Q-TOF-MS/MS - fragmentation

Introduction

Sophorae Fructus is the dry and mature fruit of Sophora japonica (L.), a leguminous plant. It has the functions of clearing away heat and toxic material, cooling blood and stopping bleeding, and is usually used for treating intestinal heat, hematochezia, nevus swelling and bleeding, dizziness, as well as red eyes.[1] It also has anticancer and estrogen-like effects, and plays a roles in prevention and treatment of cardiovascular disease, osteoporosis, and female menopause syndrome.[2] The study of the chemical components of S. japonica is of great significance for its quality control and clinical application. The main components of Sophorae Fructus are flavonoids, isoflavones, alkaloids, triterpenoid saponins, amino acids, stearic acids etc., among which isoflavones and their glycosides are the highest. However, up to now, there are few reports on the analysis of the total components of Sophorae Fructus. Sun et al identified and inferred 24 common compounds and 21 variance compounds in Sophorae Fructus from different producing areas by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS).[3] Zhang identified 131 compounds from the total extract of Sophorae Fructus in positive and negative ion modes by UPLC-Q-TOF/MS.[4] The structural types include 81 flavonoids, 18 triterpenoid saponins, 5 steroids, 2 anthraquinones, 3 phenols, and 22 others. Triterpenoid saponins were less identified, and flavonoids were mainly reported.

Traditional methods for phytochemical isolation and identification are time-consuming and labor-intensive. In recent years, techniques combining the efficient separation ability of liquid chromatography and strong identification ability of MS have been widely used in the separation and qualitative and quantitative analysis of complex Chinese medicines.[5] [6] In this study, UPLC-Q-TOF/MS was used to rapidly identify the chemical composition of Sophorae Fructus. We established a UPLC-Q-TOF/MS qualitative analysis method to analyze the constituents of Sophorae Fructus, which lay a foundation for the study of its pharmacodynamic substance basis and quality control.

At present, the studies on MS pyrolysis are relatively scattered. The electrospray ionization MS of saponin[7] [8] and flavonoid[9] [10] components has been reported. Flavonoid species can be identified according to the ultraviolet (UV) absorption characteristics of compounds and the characteristic ion fragments of the parent nuclei. But there are few literature reports on determining the connection mode between glycosyl groups in flavonoid glycosides by using the cleavage rule of MS. In this study, through the comparison of the MS data of 21 reference substances, including flavonoid oxyglycosides, flavonoid carbon glycosides, dihydro-flavonoid glycosides, isoflavone glycosides and saponins, and a large number of literature reports, we systematically deduced the cleavage characteristics of these compounds, so as to provide reference for the MS structure identification of such components.

Materials and Methods

Materials and Reagents

The prepared slices of Sophorae Fructus is obtained from Tianjin Darentang TCM Chinese Herbal Medicine Co., Ltd (Bozhou, China). Reference standards citric acid (97.0%), gallic acid (90.8%), protocatechuic acid (97.7%), protocatechuic aldehyde (99.6%), baicalein (97.9%), apigenin (99.4%), rutin (91.6%), kaempferol (93.2%), isoquercitrin (97.2%), genistein (98.8%), kaempferol-3-O-rutinoside (94.0%), kaempferol-3-O-gentiobioside (93.1%), isorhamnetin-3-O-neohesperidin (93.0%), naringin (93.5%), hesperidin (95.3%), neohesperidin (99.4%), genistin (99.9%), sophoricoside (99.6%), puerarin (95.4%), vitexin (99.1%), asperosaponin VI (94.3%), mogroside V (96.1%), ginsenoside Re (96.0%), jujuboside A (96.0%), ruscogenin (98.0%), and oleanolic acid (95.8%) were purchased from National Institute for Food and Drug Control (Beijing, China). Sophorabioside (≥98%) was purchased from Shanghai Standard Biotech Co., Ltd (Shanghai, China). Kaempferol-3-O-sophoroside (≥98%) was purchased from Chengdu Purifa Technology Development Co., Ltd. Kaempferol-3,7-di-O-glucoside (≥98%) and kaempferol-3-O-(2′′-O)-β-D-glucosyl)-β-D-rutinoside (≥98%) was purchased from Chengdu Push Biotechnology Co., Ltd (Chengdu, China). Isorhamnetin-3-O-β-D-rutinoside (≥98%) and kaempferol-3-O-β-D-sophorae-7-O-α-L-rhamnoside (≥98%) were self-made in the laboratory. Liquid chromatography-MS (LC-MS)-grade acetonitrile (ThermoFisher, United States), methanol (ThermoFisher, United States), formic acid (ThermoFisher, United States), and deionized water prepared by a Millipore Alpha-Q water purification system (Millipore, United States) were used as the mobile phase for the chromatographic separation. Other reagents were of analytical grade.

Preparation of Standards and Samples

All reference materials were dissolved in methanol and each was prepared into a solution of 0.1 mg/mL. In brief, 1.0 g of Sophorae Fructus powder (through No. 3 sieve) was accurately weighed and ultrasonicated with 30 mL 70% methanol (v/v) (250 W, 40 kHz) for 60 minutes. The sample solution and standard solution were filtered through 0.22 µm microporous filter membrane.

Instrumentation and Conditions

The UPLC-QTOF MS/MS analysis was performed using a Waters Acquity UPLC system coupled with a Xevo G2-XS QTOF mass spectrometer (Waters, United States) with an electrospray ionization ion source in MS^E mode.

The chromatographic separation process of flavonoids was performed on an ACQUITY CSH C18 (150 mm × 2.1 mm, 1.7 μm; Waters, United States) at 35°C, with a mobile phase consisting of methanol (B) and 0.05% formic acid aqueous solution (A). The gradient elution was as follows: 0–9 minutes, 10–20% eluent B; 9–27 minutes, 20–40% eluent B; 27–30 minutes, 40% eluent B; 30–39 minutes, 40–60% eluent B; 39–42 minutes, 60% eluent B; 42–48 minutes, 60–80% eluent B; 48–50 minutes, 80% eluent B; 50–50.1 minutes, 80–10% eluent B; 50.1–65 minutes,10% eluent B. The flow rate was 0.2 mL/min.

Saponins were separated by Hypersi1 Gold (100 mm × 2.1 mm, 1.9 μm; ThermoFisher Scientific, United States) at 35°C. The flow rate was 0.3 mL/min. The mobile phase was acetonitrile (B) and 0.1% formic acid (A) in water. The gradient elution was as follows: 0–1 minute, 99% eluent A; 1–5 minutes, 99–91% eluent A; 5–9 minutes, 91–84% eluent A; 9–12 minutes, 84% eluent A; 12–18 minutes, 84–67% eluent A; 18–23 minutes, 67–63% eluent A; 23–29 minutes, 63–49% eluent A; 29–34 minutes, 49–0% eluent A; 34–36 minutes, 0–99% eluent A; 36–37 minutes, 99% eluent A. The injection volume for all was 1 μL.

MS conditions were operated in both positive and negative ion modes and applied as the following: solvent gas temperature (nitrogen), 450°C; capillary voltage, 3.0/2.5 KV; an ion source temperature, 120°C; desolvation gas flow, 500 L/h; cone gas flow, 100 L/h; the low collision energy, 6 V; the high collision energy, 25 to 60 V.

Data Processing and Compound Identification

Masslynx 4.1 software (Waters, United States) was used to analyze the mass spectra peaks of Sophorae Fructus in positive and negative ion modes. According to the comparison of reference standards or references, the compounds were identified by UV spectrum, retention time, excimer ion peak, molecular formula, fragment ions, and other information combined with Scifinder database.

Results and Discussion

To systematically and qualitatively analyze the chemical components in Sophorae Fructus, the MS behavior of the existing reference standards was studied to summarize their chromatographic retention behavior, UV absorption, cracking rule, and characteristic fragment ions.

The Cracking Rules of the Deglycosylation Group of Flavone-O-diglycoside

Kaempferol-3-O-sophoroside (t _R = 26.28 minutes) and kaempferol-3-O-gentiobioside (t _R = 28.33 minutes) are isomers, their mass spectra in negative and positive ion modes are shown in [Fig. 1]. In the negative ion mode ([Fig. 1A]), kaempferol-3-O-sophoroside can obtain fragment ions of m/z 429.0819 [M – H – 162 - H₂O]^–. In the positive ion mode ([Fig. 1C]), it could generate fragment ions of m/z 449.1071 [M + H - 162]⁺, and the relative abundance (>10%) was higher than that of kaempferol-3-O-gentiobioside (<10%, [Fig. 1D]). Similarly, isorhamnetin-3-O-β-D-neohesperidoside produces fragment ions of m/z 459.0932 [M – H – 146 - H₂O]^– in the negative ion mode. The relative abundance of m/z 479.1197 [M + H - 146]⁺ fragment ion (>50%) was higher than that of isorhamnetin-3-O-β-D-rutinoside (<50%) in the positive ion mode. The retention time and ion fragments of the four reference substances showed the following regularities: (1) the polarity of flavone-O-diglycoside linked to monosaccharides in 1→2 mode was greater than that of flavone-O-diglycoside linked to monosaccharides in 1→6 mode; (2) in the negative ion mode, when the flavone-O-diglycoside is linked in 1→2 mode, it can produce [M – H – monosaccharide - H₂O]^– characteristic fragment ion, but when it is linked in 1→6 mode, it can only produce [M – H - monosaccharide]^– fragment ion, which is the same as reported in the literature[11]; (3) the relative abundance of [M + H - monosaccharide]⁺ fragment ions produced by 1→2 linkage between glycogroups is higher than that of the same fragment ions produced by 1→6 linkage between glycogroups. It is consistent with the cleavage law of Fructus aurantii flavone diglycosides in the positive ion mode reported in the literature.[12] The rules can provide a basis for identifying the most common two disaccharide connection modes (1→6, 1→2) in flavonoid oxyglycosides.

Fig. 1 Mass spectra and cleavage of (A) kaempferol-3-O-sophoroside and (B) kaempferol-3-O-gentiobioside in negative ion mode, and mass spectra of (C) kaempferol-3-O-sophoroside and (D) kaempferol-3-O-gentiobioside in positive ion mode.

The Cracking Rules of the Deglycosylation Group of Flavone-O-triglycoside

Kaempferol-3-O-β-D-sophoroside-7-O-α-L-rhamnoside (t _R = 19.66 minutes) and kaempferol-3-O-(2″-O-β-D-glucopyranosyl)-β-D-rutinoside (t _R = 25.33 minutes) are isomers. Their mass spectrometric cleavage pathways in positive and negative ion modes are shown in [Fig. 2]. In the negative ion mode, kaempferol-3-O-β-D-sophoroside-7-O-α-L-rhamnoside can obtain fragment ions of m/z 755.1894, 609.1498, 449.1126, and 284.0459, indicating that rhamnose on the C₇ position was lost first and then glucose groups on the C₃ position were lost successively. However, in the positive ion mode, the glycogroup at the end of C₃ site was lost first, and the fragment ion of m/z 595.1671 was detected. Then, after the loss of all glycogroups at the C₃ site, the rhamnose group at the C₇ site was lost, and the fragment ion with m/z of 433.1140 and 287.0568 appeared ([Fig. 2A]). Kaempferol-3-O-(2″-O-β-D-glucopyranosyl)-β-D-rutinoside in the negative ion mode could generate fragment ions of m/z 755.1838, 593.1495, 575.1411, and 284.0424. In the positive ion mode, it could generate fragment ions of m/z 779.1974, 595.1671, 493.1533, 449.1071, and 287.0568. This shows that whether in the positive or the negative ion mode, the glucose connected with 1→2 at the end was lost first, then the rhamnose connected with 1→6 was lost, and finally the glucose connected with aglycone was lost ([Fig. 2B]). Therefore, we come to the conclusion that: (1) the polarity of flavonol glycoside substitution at the C₃ and C₇ sites is greater than that of glycoside substitution at the C₃ site only. (2) Flavonol glycosides replaced by glycogroups at the C₃ and C₇ sites lose the glycogroups on the C₇ position first and then the glycogroups on the C₃ position is lost in turn in the anion mode. In the positive ion mode, the glycogroups at the end of position C₃ were lost successively, and then the glycogroups at the C₇ site were lost, which was consistent with the pyrolysis rule of flavonoids in Herba Epimedii in the positive ion mode described in the literature.[13] (3) The three monosaccharides in flavone-O-triglycoside are connected to each other. Whether in the positive or negative electrode, the glycosyl connected at the end with 1→2 is lost first, then the glycosyl connected with 1→6 is lost, and finally the loss of the glycosyl connected with aglycone.

Fig. 2 Mass fragmentation pathways deduced of (A) kaempferol-3-O-β-D-sophoroside-7-O-α-L-rhamnoside and (B) kaempferol-3-O-(2″-O-β-D-glucopyranosyl)-β-D-rutinoside in positive and negative ion modes.

Cleavage Rules of Flavonoid Carboglycosides

The mass spectrogram of puerarin in positive and negative ion modes showed fragment ions of m/z 325.0699 [M – H - 90]^–, 295.0578 [M – H - 120] ^–, 297.0750 [M + H - 120]⁺, and a series of dehydration peaks m/z 399.1077/381.0974/363.0857 were generated by the ionization peaks of [M + H]⁺ m/z 417.1158, and [M – H - 120] ^– and [M + H - 120]⁺ are the main characteristic fragments with high abundance ([Fig. 3]). Vitexin showed the same cleavage pattern, indicating that if the fragment peak of the disaccharide group does not appear first, but there are [M – H - 90]^–and [M – H - 120]^–ion fragments and [M – H - 120]^–or [M + H - 120]⁺ are the main characteristics, the fragments can basically be determined as hexacarbon flavonoid carboglycoside compounds. This is consistent with the research of Liu et al.[14] Meanwhile, according to relevant literature,[14] [15] [16] [17] in the positive ion scanning mode, the continuous dehydration of glycosyl mainly occurred, and the negative ion scanning mode has more obvious mass spectrum characteristics than the positive ion scanning mode.

Fig. 3 Secondary mass spectra of puerarin in (A) negative ion mode, (B) positive ion mode, and (C) its possible cleavage pathways.

Cleavage of Dihydroflavonoid Glycosides and Isoflavone Glycosides

Through the secondary mass spectra of naringin (t _R = 29.63 minutes), hesperidin (t _R = 30.23 minutes), and neohesperidin (t _R = 31.42 minutes) ([Table 1]), we found that: (1) the polarity of dihydroflavonoid glycosides connected in the way of 1→2 between the monosaccharides substituted on the C₇ position of dihydroflavonoid glycosides is less than that connected in the way of 1→6. (2) In the negative ion mode, in the secondary mass spectra of naringin and neohesperidin, in addition to the conventional ions [M – H - Rha]^–, [M – H – Rha - Glu]^–, there was also special ion [M – H - 120]^–with strong abundance, and the fragment ion [M – H - 162]^–could be observed. Hesperidin did not appear as these special ions. It may be due to that the rhamnose linked to the hydroxyl in the C₂ position of the glucose at the end of aglycone has rearranged and cleaved and the ion [M – H - 120]^–resulted from the loss of a hexose residue in positions 0–3. The conclusion is to be proven by further experiments. (3) In the positive ion mode, the relative abundances of ions [M + K - Rha]⁺ and [M + K – O - Rha]⁺ were higher than those of 1→6 connected when the C₇-substituted rhamnose and glucose are 1→2 connected. In the negative ion mode, the rhamnose linked to the hydroxyl in the C₂ position of the glucose at the end of aglycone is more likely to rearrange, which may be related to the different charge distribution in the positive and negative ion modes. In the positive ion mode, the charge is mainly concentrated on the added sodium ions; and in the negative ion mode, the charge is mainly distributed in the whole sugar chain.[18]

Table 1 MS² data of naringin, neohesperidin, hesperidin, and the relative abundance (%) of ions

Compound	MS2 (ESI−)	MS2 (ESI+)
Naringin	579.1885 [M – H]– 459.1290 [M – H – 120]–(100) 433.1394 [M – H – Rha]– (17.9) 415.1147 [M – H – 164]–(24.6) 271.0694 [M – H – Rha – Glu]–	619.1328 [M + K]+ (23.2) 603.1697 [M + Na]+ (100) 473.0746 [M + K – Rha]+ (4.1) 457.1020 [M + K – O – Rha]+ (3.3) 273.0744 [M + H – Rha – Glu]+ (39.8)
Neohesperidin	609.1806 [M – H]– 489.1284 [M – H – 120]–(100) 463.1176 [M – H – Rha]– (6.6) 449.0977 [M – H – 160]–(89.3) 447.0865 [M – H – 162]–(9.4) 301.0676 [M – H – Rha – Glu]–	649.1487 [M + K]+ (18.7) 633.1815 [M + Na]+ (100) 503.0856 [M + K – Rha]+ (6.6) 487.1154 [M + K – O – Rha]+ (2.0) 303.0862 [M + H – Rha – Glu]+ (34.7)
Hesperidin	609.1806 [M – H]– 463.1176 [M – H – Rha]– 301.0711 [M – H – Rha – Glu]–	649.1539 [M + K]+ (21.6) 633.1815 [M + Na]+ (100) 503.0810 [M + K – Rha]+ (2.6) 487.1064 [M + K – O – Rha]+ (1.4) 303.0862 [M + H – Rha – Glu]+ (22.1)

By comparing genistin (t _R = 24.04 minutes), sophoricoside (t _R = 28.72 minutes), and sophorabioside (t _R = 30.28 minutes), we found that the polarity of isoflavone glycosyl substitution on the C₇ position is greater than that at position C_4' ([Fig. 4]). The glycosyl group of genistein substituted at position C₇ only lost 120 fragment ions at the negative electrode. Both in the positive and negative electrodes, the glycosyl groups of sophoricoside and sophorabioside substituted at position C_4' detected the loss of 120 fragment signal, and the positive signal intensity is higher. However, whether the lost fragment signal (120 U) can be used as the diagnostic fragment of isoflavone glycosides needs further research.

Fig. 4 Secondary mass spectra of genistein and sophoricoside.

Mass Spectrometric Cleavage of Saponins

Full scan and mass spectrometric cleavage analysis were performed for saponin standard under positive and negative ion modes. The analysis results of characteristic fragments are shown in [Table 2], and the mass spectrometric cleavage pathway of asperosaponin VI is shown in [Fig. 5]. The summary rules are as follows: (1) In the negative ion mode, the saponin parent nucleus fragments are not obvious, mainly the deglycosylated fragments and [M + Cl]^–, [M – H]^– excimer ion peaks; in the positive ion mode, a series of dehydrated fragments and [M + Na]⁺ excimer ion peaks in the mother nucleus were mainly detected, while the response of deglycosylated fragments was weak. (2) The glycosyl group at C₃ position in asperosaponin VI, mogroside V, and ginsenoside Re is the last to fall off. Asperosaponin VI first lost the glycosylation at position C₂₈, ginsenoside Re first lost the glycosylation at position C₂₀, and mogroside V first lost the glycosylation at position C₂₃. This may be due to the ester bond and the ether bond on the straight chain is easier to break than the ether bond on the C₃ ring. (3) In the positive ion mode, the dehydration reaction of saponin parent nucleus fragments is only related to the number of hydroxyl groups carried on the mother nucleus, not related to the type of saponin, sugar chain and substitution position, and the number of dehydrated fragments is consistent with the number of hydroxyl groups on the mother nucleus, which is consistent with the literature report.[19] (4) Saponin aglycone: triterpenoid saponin oleanolic acid has a [M – H]^– excimer ion peak in the negative ion mode, while steroidal saponin ruscogenin has a [M + H]⁺ excimer ion peak in the positive ion mode.

Fig. 5 Mass fragmentation pathways deduced of asperosaponin VI.

Table 2 ESI-MS cleavage characteristics of six saponins

Compounds	Formula	RT (min)	Mr	m/z (ESI–)	m/z (ESI+)
Asperosaponin VI	C47H76O18	18.64	929.1000	963.4807 [M + Cl]– 927.5045 [M – H]– 603.3326 [M – H – Glc – Glc]– 471.3488 [M – H – Glc – Glc – Ara]–	952.5076 [M + Na]+ 455.3562 [M + H – Glc – Glc – Ara – H2O]+ 437.3417 [M + H – Glc – Glc – Ara – 2H2O]+ 409.3485 [M + H – 2Glc – Ara – 2H2O – CO]+ 391.3341 [M + H – 2Glc – Ara – 2H2O – CO – H2O]+
Mogroside V	C60H102O29	15.78	1,287.4300	1,331.7566 [M + HCOOH]– 1,321.6241 [M + Cl]– 1,285.6539 [M – H]– 1,123.5906 [M – H – Glc]– 961.5374 [M – H – 2Glc]– 799.4830 [M – H – 3Glc]– 637.4334 [M – H – 4Glc]– 485.1541 [Glc + Glc + Glc – H]–	1,309.6475 [M + Na]+ 1,125.6078 [M + H – Glc]+ 963.5576 [M + H – 2Glc]+ 459.3859 [M + H – 5Glc – H2O]+ 441.3770 [M + H – 5Glc – 2H2O]+ 423.3704 [M + H – 5Glc – 3H2O]+ 405.3514 [M + H – 5Glc – 4H2O]+
Ginsenoside Re	C48H82O18	15.93	947.1500	981.5267 [M + Cl]– 945.5445 [M – H]– 783.4909 [M – H – Glc]– 765.4784 [M – H – Glc – H2O]– 637.4334 [M – H – Glc – Rha]– 619.4224 [M – H – Glc – Rha – H2O]– 475.3793 [M – H – Glc – Rha – Glc]–	969.5428 [M + Na]+ 789.4763 [M + Na – Glc – H2O]+ 459.3815 [M + H – Glc – Ara – Glc – H2O]+ 441.3770 [M + H – Glc – Ara – Glc – 2H2O]+ 423.3746 [M + H – Glc – Ara – Glc – 3H2O]+ 405.3556 [M + H – Glc – Ara – Glc – 4H2O]+
Jujuboside A	C58H94O26	19.29	1,207.3500	1,241.5747 [M + Cl]– 1,206.6047 [M – H]– 1,074.5632 [M – H – Ara]– 911.5027 [M – H – Ara – Glc]– 749.4507 [M – H – Ara – 2Glc]– 603.3880 [M – H – Ara – 2Glc – Rha]– 471.3444 [M – H – Ara – 2Glc – Rha – Ara]–	1,229.5935 [M + Na]+ 455.3650 [M + H – Ara – 2Glc – Rha – Ara – H2O]+ 437.3417 [M + H – Ara – 2Glc – Rha – Ara – 2H2O]+
Ruscogenin	C27H42O4	30.84	430.6300	311.1677 [M – H – 118]–	431.3171 [M + H]+ 413.3069 [M + H – H2O]+ 395.3064 [M + H – 2H2O]+
Oleanolic acid	C30H48O3	32.65	456.3600	455.7130 [M – H]–	439.3678 [M + H – H2O]+ 411.3676 [M + H – H2O – CO]+ 393.3522 [M + H – H2O – CO – H2O]+

Identification of Chemical Constituents of Sophorae Fructus

Identification of Flavonoids

When analyzed the test sample according to chromatographic conditions (1), under the negative ([Fig. 6A]) and positive ([Fig. 6B]) ion scanning modes, the separation degree and ionization degree of each component of Sophorae Fructus meet the requirements. Identification of flavonoids: first, the types of flavonoids were speculated according to the characteristics of UV spectrum. Combined with the above MS rules and literature references, 84 compounds were finally identified in positive and negative ion modes, including 51 flavonoids, 12 isoflavones, 3 dihydroflavonoids, 8 organic acids and 10 amino acids, and sugars, as shown in [Table 3].

Table 3 Analysis and identification of flavonoids from Sophorae Fructus by UPLC-QTOF mass spectrometry

No.	Component name	Formula	Calculated mass (m/z)	Measured mass (m/z)	Mass error (ppm)	RT (min)	Adducts	Fragment ions (m/z, ESI–/ESI+)
1	Arginine	C6H14N4O2	173.1044	173.1036	–4.62	1.02	[M – H]–	173.1036
2	Alanine	C3H7NO2	134.0459	134.0464	3.73	1.12	[M + HCOO]–	134.0464
3	α–Sophora	C12H22O11	387.1144	387.1134	–2.58	1.30	[M + HCOO]–	387.1134
4	Aspartic acid	C4H7NO4	132.0302	132.0296	–4.55	1.33	[M – H]–	132.0296
5	Arabinose	C5H10O5	195.0510	195.0514	2.05	1.37	[M + HCOO]–	195.0514
6	Malic acid	C4H6O5	133.0142	133.0137	–3.76	2.17	[M – H]–	133.0137
7	Citric acid[a]	C6H8O7	191.0197	191.0191	–3.14	3.42	[M – H]–	191.0191
8	Phenylalanine	C9H11NO2	164.0717	164.0710	–4.27	4.06	[M – H]–	164.0170
9	Gallic acid[a]	C7H6O5	171.0288	171.0287	–0.58	4.11	[M + H]–	171.0287, 125.0300
10	γ-Glutamyltyrosine	C14H18N2O6	309.1092	309.1088	–1.29	6.32	[M – H]–	309.1088
11	Tryptophan	C11H12N2O2	203.0826	203.0822	–1.97	7.16	[M – H]–	203.0822
12	Kaempferol-3-O-sophoroside-7-O-glucoside[b]	C33H40O21	771.1989	771.1990	0.13	8.83	[M – H]–	771.1990, 593.1495, 447.089, 285.0403
13	Protocatechuic acid[a]	C7H6O4	153.0193	153.0191	–1.31	8.97	[M – H]–	153.0191
14	Methyl gallate	C8H8O5	183.0299	183.0296	–1.64	10.13	[M – H]–	183.0296, 169.0179, 125.0219
15	4-(β-D-Glucopyranosyloxy)-3-hydroxyphenyl caffeate[b]	C21H22O11	449.1089	449.1083	–1.34	10.76	[M – H]–	449.1083, 287.0553
16	Protocatechualdehyde[a]	C7H6O3	183.0299	183.0301	1.09	10.86	[M + HCOO]–	183.0301
17	1,6-di-O-galloyl-β-D-glucose	C20H20O14	483.0780	483.0791	2.28	11.49	[M – H]–	483.0791, 331.0651, 313.0550, 169.0130, 125.0235
18	Kaempferol-3-O-gentionbioside-7-O-glucoside[b]	C33H40O21	771.1989	771.1990	0.13	12.29	[M – H]–	771.1990, 609.1448, 447.0890, 285.0369
19	Kaempferol-3-O-(2″-O-β-D- glucopyranosyl)-β-D-rutinoside-7-O-glucoside[b]	C39H50O25	917.2568	917.2574	0.65	12.47	[M – H]–	917.2574, 755.2006, 577.1556, 446.0854, 284.0320
20	Lepidoside	C26H28O14	563.1406	563.1374	–5.68	14.09	[M – H]–	563.1374, 419.1205, 285.0403
21	Genistein-7-O-β-D-glucoside 4′-O-sophoroside	C33H40O20	757.2186	757.2182	–0.53	14.45	[M + H]+	757.2182, 595.1622, 433.1140, 271.0597
22	Genistein-7,4′-di-O-β-D-glucoside	C27H30O15	595.1657	595.1664	1.18	14.91	[M + H]+	595.1664, 433.1140, 271.0597
23	Quercetin-3-O-(3′′′-O-β-D- glucopyranosyl)-β-D-neohesperidoside[b]	C33H40O21	771.1989	771.1990	0.13	15.59	[M – H]–	771.1990, 609.1448, 462.0806, 431.0984, 299.0180
24	Apigenin-7,4′-diglucoside	C27H30O15	595.1657	595.1671	2.35	15.88	[M + H]+	595.1671, 433.1097, 271.0630
25	Neoeriocitrin[b]	C27H32O15	595.1668	595.1653	–2.52	16.08	[M – H]–	595.1653, 431.0984, 287.0553
26	Diosmetin-7-O-sophoroside[b]	C28H32O16	669.1672	669.1663	–1.35	16.33	[M + HCOO]–	623.1638, 461.1086, 445.0786, 268.0387
27	Quercetin-3-O-(2″-O-β-D- glucopyranosyl)-β-D-rutinoside-7-O-rhamnoside[b]	C39H50O25	917.2568	917.2533	–3.82	17.30	[M – H]–	917.2533, 771.1990, 609.1397, 461.1306, 301.0356
28	Genistein-7-O-β-D-glucoside-4′-O-neohesperidoside	C33H40O19	739.2091	739.2086	–0.68	17.88	[M – H]–	739.2086, 577.1654, 431.0984, 415.1070, 268.0454
29	Kaempferol-3,7-diglucoside[a]	C27H30O16	609.1461	609.1448	–2.13	18.15	[M – H]–	609.1448, 447.0977, 285.0403
30	Kaempferol-3-O-(4′′′-O-β-D- glucopyranosyl)-β-D-rutinoside[b]	C33H40O20	755.2040	755.2062	2.91	18.74	[M – H]–	755.2062, 593.1545, 446.0854, 285.0403
31	Apigenin-7-O-(2′′-O-sophorosyl)-β-D-rutinoside[b]	C39H50O24	903.2765	903.2797	3.54	18.90	[M + H]+	903.2797, 741.2186, 579.1738, 433.1140, 271.0597
32	Ternatumoside VIII[b]	C39H50O24	901.2619	901.2601	–2.00	19.29	[M – H]–	901.2601, 755.2062, 593.1495, 430.0917, 284.0355
33	Kaempferol-3-O-sophoroside-7-O-rhamnoside[a]	C33H40O20	757.2186	757.2196	1.32	19.64	[M + H]+	757.2196, 595.1671, 433.1140, 287.0568
34	Isorhamnetin-3-O-(4′′-O-rutinosyl)-β-D-rutinoside[b]	C40H52O25	933.2870	933.2865	–0.54	20.12	[M + H]+	933.2865, 787.2250, 625.1698, 463.1243, 317.0664
35	Isorhamnetin-3-O-sophoroside-7-O-rhamnoside[b]	C34H42O21	785.2146	785.2151	0.64	20.72	[M – H]–	785.2151, 639.1575, 459.1283, 314.0445
36	Kaempferol-3-O-glucoside-7-O-rutinoside[b]	C33H40O20	755.2040	755.2062	2.91	21.03	[M – H]–	755.2062, 635.0911, 608.1398, 447.0933, 285.0403
37	Dihydrokaempferol 3-O-glucoside	C21H22O11	449.1089	449.1083	–1.34	21.45	[M – H]–	449.1083, 287.0578
38	Genistein-7-O-malonylglucoside-4′-O-glucoside	C30H32O18	681.1661	681.1641	–2.94	21.82	[M + H]+	681.1641, 433.1140, 271.0597
39	Quercetin-3-O-β-D-glucopyranosyl(1→2)-[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside	C33H40O21	773.2135	773.2154	2.46	22.12	[M + H]+	773.2154, 611.1609, 465.1011, 303.0498
40	Quercetin 3-O-gentiobioside	C27H30O17	627.1556	627.1563	1.12	23.08	[M + H]+	627.1563, 465.1033, 303.0494
41	Quercetin-3-O-(6′′′-O-adipoyl)-β-D-rutinoside[b]	C33H38O19	739.2080	739.2090	1.35	23.21	[M + H]+	739.2090, 465.1033, 303.0494
42	Quercetin-3-O-(6″-O-(3′′′-O-arabinose)-α-L-rhamnosyl)-β-D-neohesperidoside[b]	C38H48O24	887.2463	887.2480	1.92	23.46	[M – H]–	887.2480, 741.1857, 609.1448, 301.0356
43	Compactin [b]	C27H30O16	611.1607	611.1609	0.33	23.67	[M + H]+	611.1609, 449.1079, 287.0559
44	Genistin[a]	C21H20O10	431.0984	431.0984	0	24.03	[M – H]–	431.0984, 269.0551
45	Apigenin-7-O-(3′′′-O-acetyl)-β-D-rutinoside	C29H32O15	619.1668	619.1690	3.55	24.85	[M – H]–	619.1690, 431.0984, 268.0387
46	Kaempferol-3-O-(2″-O-β-D- glucopyranosyl)-β-D-rutinoside[a]	C33H40O20	757.2186	757.2182	–0.53	25.04	[M + H]+	757.2182, 595.1671, 449.1071, 287.0672
47	Rhamnocitrin-3-O-β-D-glucopyranosyl(1→2)-D-Apio-α-D-furanoside -4′-O-glucoside	C33H40O20	757.2186	757.2182	–0.53	25.31	[M + H]+	757.2182, 595.1622, 463.1243, 301.0723
48	Isorhamnetin-3-O-sophoroside	C28H32O17	641.1712	641.1703	–1.40	25.94	[M + H]+	641.1703, 479.1197, 317.0664
49	Kaempferol-3-O-sophoroside[a]	C27H30O16	609.1461	609.1448	–2.13	26.12	[M – H]–	609.1448, 429.0819, 285.0507
50	Multiflorin B	C27H30O15	593.1512	593.1495	–2.87	26.58	[M – H]–	593.1495, 431.0984, 284.0320
51	Naringin[a]	C27H32O14	579.1719	579.1736	2.94	27.05	[M – H]–	579.1736, 433.1239, 271.0601
52	Kaempferol-3-O-α-L-rhamnopyranosyl(1→4)]-β-D-glucopyranoside	C27H30O15	593.1512	593.1495	–2.87	27.73	[M – H]–	593.1495, 447.0933, 429.0776, 285.0403
53	6″-β-D-Xylosegenistin	C26H28O14	587.1377	587.1366	–1.87	28.05	[M + Na]+	587.1366, 433.1181, 271.0609
54	Rutin[a]	C27H30O16	633.1432	633.1448	2.53	28.16	[M + Na]+	633.1448, 465.1033, 303.0565
55	Isoquercitrin[a]	C21H20O12	463.0882	463.0902	4.32	28.59	[M – H]–	463.0902, 300.0323
56	Kaempferide-3-O-glucoside	C22H22O11	463.1235	463.1243	1.73	28.66	[M + H]+	463.1243, 301.0723
57	Sophoricoside[a]	C21H20O10	433.1129	433.1140	2.54	28.74	[M + H]+	433.1140, 271.0630
58	Helieianeoside A[b]	C32H38O19	725.1935	725.1947	1.66	30.06	[M – H]–	725.1947, 593.1495, 431.0984, 285.0403
59	Sophorobioside[a]	C27H30O14	601.1533	601.1518	–2.50	30.28	[M + Na]+	601.1518, 579.1738, 433.1140, 271.0597
60	Apigenin-7-O-neohesperidoside	C27H30O14	577.1563	577.1556	–1.21	30.83	[M – H]–	577.1654, 431.0984, 413.0867, 269.0450
61	Apigenin-7-O-rutinoside	C27H30O14	577.1563	577.1556	–1.21	31.43	[M – H]–	577.1556, 431.0984, 269.0450
62	Apigenin-4′-O-rutinoside[b]	C27H30O14	577.1563	577.1556	–1.21	31.81	[M – H]–	577.1556, 431.0984, 268.0387
63	Kaempferol-3-O-rutinoside[a]	C27H30O15	595.1657	595.1671	2.35	32.62	[M + H]+	595.1721, 449.1071, 287.0603
64	Apigenin-7-O-gentiobioside	C27H30O15	593.1512	593.1495	–2.87	33.02	[M – H]–	593.1495, 447.0933, 269.0450
65	Isorhamnetin-3-O-β-D-rutinoside[a]	C28H32O16	625.1763	625.1749	–2.24	33.31	[M + H]+	625.1749, 479.1197, 317.0664
66	Diosmetin-7-O-glucopyranosyl-(6→1)-O-arabinopyranoside	C27H30O15	593.1512	593.1495	–2.87	33.80	[M – H]–	593.1495, 461.1102, 299.0576
67	Apigenin-7-O-[6-O-acetyl-2-O-(6-O-acetyl-β-D-glucopyranosyl)]-β-D-glucoside[b]	C31H34O17	677.1723	677.1702	–3.11	34.45	[M – H]–	677.1702, 473.1059, 268.0372
68	Apigenin-7-O-(4,6-di-O-acetyl)-β-D-glucoside-4′-O-β-D-glucoside[b]	C31H34O17	723.1778	723.1840	8.58	34.62	[M + HCOO]–	723.1840, 677.1646, 431.0989, 268.0372
69	6″-O-Acetylnaringin[b]	C29H34O15	621.1825	621.1818	–1.13	34.72	[M – H]–	621.1826, 473.1068, 271.0601
70	Genistein-4′-O-malonylglucoside	C24H22O13	519.1133	519.1116	–3.28	35.04	[M + H]+	519.1116, 271.0597
71	Kakkanin	C27H30O14	579.1708	579.1689	–3.28	35.31	[M + H]+	579.1689, 447.1278, 285.0774
72	Euryanoside[b]	C29H32O15	619.1668	619.1639	–4.68	35.48	[M – H]–	619.1639, 473.1068, 269.0450
73	Acacetin-5-O-α-L-mannopyranosyl(1→2)-α-L-rhamnopyranoside[b]	C28H32O13	575.1770	575.1411	–6.24	36.11	[M – H]–	575.1411, 431.0984, 283.0600
74	Apigenin-5-O-acetyl-7-O-neohesperidoside[b]	C29H32O15	619.1668	619.1639	–4.68	36.74	[M – H]–	619.1639, 473.1068, 311.0552, 269.0450
75	Apigenin-7-O-rhamnoside	C21H20O9	415.1035	415.1028	–1.69	37.22	[M – H]–	415.1028, 268.0387
76	Acacetin-7-(6-malonylglucoside)	C25H24O13	555.1115	555.1107	–1.44	37.72	[M + Na]+	555.1107, 285.0729
77	Apigenin-7,4′-di-O-rhamnoside[b]	C27H30O13	561.1614	561.1620	1.07	38.12	[M – H]–	561.1620, 415.1063, 268.0400
78	Genistein[a]	C15H10O5	271.0601	271.0597	–1.48	38.46	[M + H]+	271.0601
79	Apigenin-7-O-α-L-mannopyranosyl (1→3)-α-L-rhamnopyranoside[b]	C27H30O13	561.1614	561.1620	1.07	38.58	[M – H]–	561.1692, 415.1063, 397.0893, 269.0484
80	Diosmetin	C16H12O6	299.0561	299.0570	3.01	39.69	[M – H]–	299.0570, 284.0320, 255.0295
81	Acacetin	C16H12O5	283.0612	283.0600	–4.24	40.36	[M – H]–	283.0600, 255.0295, 242.9433
82	Kaempferol[a]	C15H10O6	287.0550	287.0559	3.14	40.71	[M + H]+	287.0559
83	Baicalein[a]	C15H10O5	269.0455	269.0450	–1.86	41.11	[M – H]–	269.0450
84	Apigenin[a]	C15H10O5	269.0455	269.0450	–1.86	41.41	[M – H]–	269.0450

^a Compared with reference substance.

^b Be first found in Sophora.

Fig. 6 Total ion flow diagram of Sophorae Fructus components in (A, C) negative ion mode and (B, D) positive ion mode.

Among the flavonoids, flavonols are the most abundant. Take compound 35 in [Table 3] as an example to illustrate the identification process. According to the UV maxima at 260 and 353 nm, the structural type was flavonol with substitution at the C₃ position. In the negative ion mode, the MS² of the aglycon-related ions were m/z 315.0502, 300.0277, 284.0320, 269.0450, 255.0295, 243.0678, 215.0729, 125.0258, which is identified as isorhamnetin by comparison with the literature.[20] Compound 35 displayed a [M – H]^– ion at m/z 785.2151 and ions at m/z 639.1575 [M – H - Rha]^–, m/z 459.1283 [M – H – Rha – Glu - H₂O]^–, as well as m/z 315.0502 [M – H - Rha - 2Glu]^–. It was noted the presence of the m/z 459.1283 ion, which results from the loss of a hexose (180 u). This H₂O loss shows that the dihexosyl should have a 1→2 interglycosidic linkage because, as referred to above when the 1→2 bond versus the 1→6 bond was compared, the 1→6 bond is difficult to break. In the positive ion mode, compound 35 produced [M + H]⁺ ion at m/z 787.2308 and in its MS² fragmentation of these ions (m/z 625.1698 [M + H – Glu]⁺→m/z 463.1243 [M + H – 2Glu]⁺→m/z 317.0664 [M + H – 2Glu – Rha]⁺) can be observed. Combined with the above flavone cleavage rules, rhamnose was bound to a phenolic hydroxyl at position C₇, dihexosides with interglycosidic linkage 1→2 was substituted at position C₃. Therefore, compound 35 was proposed to be isorhamnetin-3-O-sophoroside-7-O-rhamnoside by comparison with the literature.[11]

Identification of Saponins

After analyzing the test sample according to chromatographic conditions (2), the total ion flow diagram is shown in [Fig. 6C] (negative ion scanning mode) and [Fig. 6D] (positive ion scanning mode). A total of 58 compounds were identified by using the above saponin cleavage rules in combination with relevant literature and reference standards, including 39 saponins, 10 phenolic acids, 3 fatty acids, 2 phenylpropanoids, 1 flavonol, and 3 others ([Table 4]).

Table 4 Analysis and identification of saponins from Sophorae Fructus by UPLC-Q-TOF mass spectrometry

No.	Component name	Formula	Calculated mass (m/z)	Measured mass (m/z)	Mass error (ppm)	RT (min)	Adducts	Fragment ions (m/z, ESI–/ESI+)
85	Gallic acid-O-glucoside (isomer)	C13H6O10	331.0671	331.0687	4.83	1.51	[M – H]–	331.0761, 169.0122, 125.0242
86	Gallic acid-O-diglucoside	C19H26O15	517.1169	517.1176	1.35	2.33	[M + Na]+	517.1176, 355.0602, 171.0284, 125.0230
87	p-Hydroxybenzoic acid glucoside	C13H16O8	299.0772	299.0756	–5.35	2.67	[M – H]–	299.0756, 137.0223
88	M-digallic acid	C12H16O6	321.0252	321.0264	3.74	2.86	[M – H]–	321.0264, 169.0122, 125.0219
89	1-p-anisate-glucopyranuronic acid[a]	C14H16O9	328.0794	328.1377	8.23	3.11	[M + e]+	328.1377, 279.0945, 141.9588
90	Gallic acid-O-glucoside (isomer)	C13H6O10	331.0671	331.0687	4.83	3.23	[M – H]–	331.0761, 169.0122, 125.0242
91	Digallic acid[a]	C14H12O9	323.0409	323.0394	–4.64	3.37	[M – H]–	323.0394, 169.0122, 125.0242
92	3,5-Dihydroxy-4-[(3,4,5-trihydroxybenzoyl)oxy]benzoic acid	C14H10O9	321.0252	321.0264	3.74	3.52	[M – H]–	321.0264, 178.9772, 144.0444
93	Amygdalinic acid[a]	C20H28O13	475.1457	475.1428	–6.11	3.67	[M – H]–	475.1428, 329.0865, 313.0917, 268.0372, 151.0381
94	2,3-Dihydroxy-3-(3,4,5-trimethoxyphenyl)propyl-β-D-glucopyranoside[a]	C18H28O11	419.1559	419.1549	–2.39	3.77	[M + Na]+	443.1170, 421.1345, 289.0913, 158.9624, 127.0396
95	Everlastoside H[a]	C21H28O14	503.1406	503.1408	0.40	3.92	[M – H]–	503.1408, 345.0841, 323.1351, 178.9772, 145.9299
96	2-Hydroxy-1-(hydroxymethyl)-2-(3,4,5-trimethoxyphenyl)ethyl-β-D-glucopyranoside[a]	C18H28O11	465.1614	465.1653	8.39	4.03	[M + HCOO]–	465.1225, 419.1172, 315.0724, 235.9240, 178.9772
97	Pothobanoside C[a]	C26H38O16	651.2142	651.2100	–6.45	4.24	[M + HCOO]–	651.2100, 443.1891, 329.0865, 153.0169, 137.0223
98	Tuberosinine D	C19H28O12	471.1478	471.1494	3.40	4.43	[M + Na]+	471.1494, 343.0466, 315.0544, 153.0164
99	Di-O-galloyl glucose	C20H19O14	483.0780	483.0778	–0.41	4.61	[M – H]–	483.0778, 331.0612, 271.0450, 243.0513, 169.0148
100	Isorhamnetin	C16H12O7	315.0510	315.0506	–1.27	17.49	[M – H]–	315.0506, 300.0286, 285.0383, 269.0436
101	28–(Hydroxymethyl)olean-12-en-3β-yl-O-β-D-glucopyranosyl-(1→6)-O-β-D-galactopyranosyl(1→4)-O-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranosiduronic acid[a]	C55H90O22	1,101.5851	1,101.5781	–6.36	17.56	[M – H]–	1101.5781, 957.5237, 795.4545, 633.3990, 458.3735, 439.3592, 421.3480
102	3-[(O-hexopyranosyl-(1→2)-O-hexopyranosyl-(1→3)-O-hexopyranuronosyl-(1→4)-6-deoxyhexopyranosyl)oxy]-, 21-acetate 22-(2-methylpropanoate), (3β)-Olean-12-ene-15,16,21,22,28-pentol[a]	C60H96O28	1,287.5986	1,287.5944	–3.26	17.66	[M + Na]+	1287.5944, 1265.6073, 1103.5602, 765.4705, 619.1520, 499.1179, 471.0606, 458.3761, 445.1229, 417.1301, 401.2097
103	Polybosaponin A	C48H76O19	979.4878	979.4835	–4.39	18.86	[M + Na]+	979.4835, 811.4466, 649.3914, 473.3636, 455.3542, 437.3432
104	Azukisaponin V	C48H78O18	941.5115	941.5151	3.83	19.01	[M – H]–	941.5151, 795.4539, 615.3890, 457.3663, 441.3744, 423.3672, 405.3513
105	Azukisaponin II	C42H68O14	795.4536	795.4539	0.38	19.33	[M – H]–	795.4539, 633.1541, 617.4020, 441.3744, 423.3627, 405.3471, 395.0494
106	Dehydrosoyasaponin I	C48H76O18	941.5104	941.5088	–1.70	19.67	[M + H]+	941.5088, 795.4539, 633.3990, 457.3667, 439.3549, 421.3438, 403.3315, 395.0746
107	Abrisaponin I	C48H74O20	971.4846	971.4894	4.94	19.87	[M + H]+	971.4894, 825.4259, 649.4018, 469.3304, 451.3204, 433.1133, 405.3430
108	Umbellatoside A[a]	C48H76O19	979.4878	979.4835	–4.39	19.96	[M + Na]+	979.4835, 819.4411, 649.3965, 473.3636, 455.3542, 437.3389, 421.3444, 391.1188
109	Astragaloside VIII	C47H76O17	935.4980	935.4990	1.07	20.23	[M + Na]+	935.4990, 789.4240, 633.1387, 441.3744, 423.3627, 405.3513
110	Putranoside C	C47H72O19	963.4929	963.4963	3.53	20.65	[M + Na]+	963.4963, 779.4532, 633.4012, 457.3692, 439.3565, 421.3480, 391.1228
111	Yunganoside D1 [a]	C48H74O19	977.4722	977.4739	1.74	21.03	[M + Na]+	977.4739, 831.4089, 633.4012, 453.3394, 435.3260
112	Soyasaponin Bg	C47H74O17	933.4824	933..4748	–8.15	21.08	[M + Na]+	933.4788, 765.4444, 633.4012, 455.3455, 439.3565
113	(3β,4β)-23-hydroxy-22-oxoolean-12-en-3-yl-O-6-deoxy-α-L-mannopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosiduronic acid	C48H76O18	963.4929	963.4900	–3.01	21.14	[M + Na]+	963.4900, 817.4276, 633.4012, 439.3565, 421.3486
114	Soybean phenol A	C42H66O14	793.4380	793.4404	3.03	21.25	[M – H]–	793.4404, 631.3856, 455.0141
115	Glycyrrhetinic acid-3-O-Glucopyranosiduronic acid-29-O-glucoside	C42H64O15	831.4143	831.4148	0.61	21.62	[M + Na]+	831.4148, 647.4064, 471.3449, 453.3351, 435.3260, 424.1924, 407.3350
116	Uralsaponin X	C50H74O22	1,049.4569	1,049.4613	4.19	21.81	[M + Na]+	1049.4613, 741.3734, 704.2580, 565.3466, 525.1416, 507.1360, 481.0825, 457.2379, 439.3565, 421.3444, 403.3354
117	(3β,4β,22β)-22,23-Dihydroxy-11-oxoolean-12-en-3-yl-O-6-deoxy-α-L-mannopyranosyl-(1→2)-O-β-D-galactopyranosyl-(1→2)-β-D-glucopyranosiduronic acid	C48H76O19	979.4878	979.4835	–4.39	22.23	[M + Na]+	979.4835, 833.4841, 671.3387, 455.3542, 445.1306, 423.3585, 409.1622
118	Wistariasaponin D	C47H74O17	909.4853	909.4829	–2.64	22.46	[M – H]–	909.4829, 763.4296, 631.3805, 455.3504
119	Glycyrflavoside B	C47H72O18	947.4616	947.4581	–3.70	22.95	[M + Na]+	947.4581, 801.3914, 669.3496, 471.3449, 453.3351, 435.3260
120	(3β,4α,22β)-22,23-dihydroxyolean-12-en-3-yl-O-β-D-arabinofuranosyl-(1→2)-O-6-deoxy-β-D-galactopyranosyl-(1→2)-β-D-glucopyranosiduronic acid	C47H76O17	935.4980	935.4927	–5.67	23.84	[M + Na]+	935.4927, 781.4766, 635.3337, 441.3658, 423.3585, 405.3471
121	Soyasaponin I	C48H78O18	943.5261	943.5261	0	23.99	[M + H]+	943.5261, 797.4693, 635.4162, 441.3781, 423.3627
122	Soyasaponin III	C42H68O14	795.4536	795.4539	0.38	24.69	[M – H]–	795.4539, 633.3983, 455.3504
123	Kaikasaponin III	C48H78O17	949.5137	949.5123	–1.48	25.42	[M + Na]+	949.5123, 803.4489, 601.4117, 425.3773, 407.3681
124	β-D-glucopyranosiduronic acid deriv-oleanane	C48H78O17	949.5137	949.5123	-1.48	25.87	[M + Na]+	949.5123, 803.4431, 657.3563, 633.4012, 457.3648, 437.1935, 425.3773, 407.3681
125	Pisumsaponin II	C48H76O18	939.4959	939.4937	–2.34	25.97	[M – H]–	939.4937, 793.4346, 631.3856, 455.3504
126	Kaikasaponin I	C42H68O13	803.4558	803.4547	–1.37	26.33	[M + Na]+	803.4547, 641.2833, 425.3773, 407.3681
127	Azukisaponin I	C42H68O13	779.4587	779.4567	–2.57	26.65	[M – H]–	779.4567, 617.4016, 441.3724
128	Kakkasaponin I	C47H76O16	895.5061	895.5054	–0.78	26.92	[M – H]–	895.5054, 749.4451, 599.3972, 441.3724
129	Phaseoside IV	C48H76O17	947.4980	947.4958	–2.32	27.09	[M + Na]+	947.4958, 803.4025, 641.4025, 617.4020, 441.3744, 423.3627, 405.3513
130	Kakkasaponin II	C42H66O13	777.4431	777.4463	4.12	27.91	[M – H]–	777.4463, 615.3941, 437.3435
131	Zygophyloside M[a]	C40H62O13	795.4536	795.4539	0.38	28.16	[M + HCOO]–	795.4539, 749.4507, 633.3983, 441.3724
132	Kakkasaponin III	C47H74O16	917.4875	917.4889	1.53	28.39	[M + Na]+	917.4889, 749.4507, 617.4071, 441.3701, 423.3627, 405.3471
133	Paradoxoside E[a]	C37H56O11	699.3720	699.3745	3.58	28.64	[M + Na]+	699.3582, 471, 453.1652, 437.2020, 407.3681
134	Presenegenin	C30H46O7	563.3226	563.3226	0	29.44	[M + HCOO]–	563.3226, 502.2917, 311.1677, 265.1462
135	Paritriside C[a]	C41H64O12	771.4295	771.4313	2.33	29.59	[M + Na]+	771.4313, 609.3352, 441.3701, 423.3627, 405.3471
136	Aspacochinoside O[a]	C33H52O12	641.3532	641.3506	–4.06	29.95	[M + H]+	641.3506, 479.2936, 317.1825, 301.0718, 279.2312
137	Coronaric acid	C18H32O3	295.2279	295.2259	–6.78	30.31	[M – H]–	295.2259, 265.1462
138	Δ4-Pregnen-20β-ol-3-one glucoside[a]	C27H42O7	479.3003	479.2981	–4.59	30.40	[M + H]+	479.2981, 318.2985, 301.0789, 281.2932
139	Oleanonic acid	C31H50O3	453.3374	453.3346	–6.18	31.03	[M – H]–	453.3316, 325.1842, 285.1703
140	Linolenic acid	C18H32O2	277.2173	277.2166	–2.53	32.56	[M – H]–	277.2166, 251.1630, 99.9244
141	Oleanolic acid[b]	C30H48O3	455.3531	455.3548	3.74	32.65	[M – H]–	455.3548, 325.1842, 271.2271
142	Methyl 9-hexadecenoate	C17H32O2	269.2475	269.2500	9.29	32.96	[M + H]+	269.2500, 255.2637, 184.0740

^a Be first found in Sophora.

^b Compared with reference substance.

Triterpenoid saponins are mainly contained in Sophorae Fructus, and the structure is mostly oleanene type. The sugar chain structure in saponins is easy to be removed during cracking. If it is a branched glycosyl group and the two terminal glycosyl groups are different, the fragment peaks that lose the two terminal glycosyl groups will appear, so it is easy to distinguish between branched glycosyl groups and straight chain glycosyl groups.[21] Compound 104 in [Table 4] is taken as an example to derive the cracking rule of these compounds. Compound 104 was detected at m/z 941.5151 [M – H]^– in the negative ion mode. The fragment ion at m/z 795.4539 indicated the loss of a deoxyhexose residue (146 u); peaks at m/z 615.3890 [aglycone + GluA – H₂O – H]^–, m/z 457.3663 [aglycone – H]^–, and m/z 483.1363 [Rha + Glu + GluA – H]^– were presented in spectra. In the positive ion mode, the characteristic fragment ions at m/z 965.5106, 819.4528, 617.4071, 441.3744, 423.3627, 405.3513 corresponded to [M + Na]⁺, [M + Na – 146]⁺, [M + H – 146–162–H₂O]⁺, [M + H – 146–162–176–H₂O]⁺, [M + H – 146–162–176–2H₂O]⁺, [M + H – 146–162–176–3H₂O]⁺, according to the above rules of saponins, there are three hydroxyl groups in the parent nucleus. Combined with Scifinder database and related literature,[22] it was speculated that the compound may be azukisaponin V.

Conclusion

In this experiment, the UPLC-Q-TOF-MS/MS method was used to quickly characterize the chemical components of Sophorae Fructus in positive and negative ion modes. The cracking rules of main flavone glycosides and saponins, which were preliminarily discussed, were helpful to improve the structural analysis efficiency and provide reference for the rapid screening and identification of flavonoids and saponins. From the data presented, 142 compounds were analyzed and inferred, including 67 flavonoids, 39 saponins, 18 organic acids, 10 amino acids and sugars, 2 phenylpropanes, 3 fatty acids, and 3 other types. A total of 43 components were first reported from the genus Sophora. This work will be helpful for the further study of pharmacodynamic material basis and quality evaluation of Sophorae Fructus.

Through the application of LC-MS technology, the repeated identification of known compounds by traditional separation and purification methods is avoided, which is conducive to saving resources, increasing the discovery probability of new compounds, and effectively improving work efficiency. It provides ideas and methods for the basic research and new drug development of traditional Chinese medicine and other complex substrates.

Conflicts of Interests

None declared.

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Publication History

Article published online:
15 August 2022